Biochemical process for production of vitamins of the b complex



reams Dec. so, 1049 UNITED STAT ES PAT BIOCHEMICAL PROCESS FOR PRODUCTIOM F VITAMINS OF THE B COMPLEX 'Jerome Martin. Terre Haute, Ind., assignor to Commercial No Drawing.

My invention relates to the production of vitamins of the B complex. More particularly, it is concerned with the synthesis of riboflavin and other related vitamins, such as pantothenic acid, by the action of the fungus Eremothecium Ashbrii on certain solid substrates.

It has previously been known that the organism Eremothecium Ashbuii is capable of synthesizing riboflavin in limited quantities from gelatinous media of the nature of agar preparations and the like containing carbohydrate and protein. These'media contained glucose as the carbohydrate source and peptone and beef bullion as the protein source. The yields of riboflavin thus produced, however, were relatively small and in general it required approximately three weeks or more to secure such yields. With liquid media, utilizing the peptone-glucose typ nutrients referred to above, the yields of riboflavin were inferior to those obtained where the gelatinous or so-called semi-solid media were utilized. More recently a process for producing such vitamins has been discovered which involves subjecting mashes consisting essentially of protein and carbohydrate to the fermenting action of an active culture of Eremothecium Ashbyii while aerating the mash throughout the fermentation period. This procedure is described in detail in copending application, U. S. Serial No. 434,901, filed March 18, 1942, by H. R. Stiles, Patent No. 2,483,855. Such a process is readily adaptable to commercial production of riboflavin and pantothenic acid by fermenting large volumes of liquid media.

The aerated liquid process, however, while producing improved yields over the older methods, requires large quantities of air for aeration there of, usually involving the necessity for a high pressure air system. Furthermore, for certain purposes, for example in cases where the riboflavin material is to be used as a supplement for animal feeds and is to be blended therewith, the use of the liquid fermentation process presents the dimculties and expense of evaporation, drying and uniform blending with the particular feed materials and nutrients.

It is therefore an object of my invention to provide a process for preparing a riboflavin material which requires a minimum of air to supply the necessary aerobic conditions for the adequate solvents Corp ration, 1116., a corporation of Maryland Application December 6, 1944, Serial No. 566,957

1 Claim. (Cl. 195-87) Terre Haute,

Other objects will appear hereinafter.

These and other objects are accomplished by carrier, with the addition of a nutrient solution.

80 longer periods may be required for larger batches.

The solid base or carrier may itself furnish a major portion of the nutrient necessary for the growth and vitamin production of the organism. The necessary nutrients may be added in an appropriate form such as by a liquid nutrient solution used to moisten and thinly coat the carrier particles.

The process of my invention requires the selection of a suitable base or support for the organism. The nutrient solution is added to the base in a sufficient quantity to support the normal growth of. said fungus, the base and nutrient solution being thoroughly mixed to insure a complete and uniform distribution of the nutrient solution upon the base. The mixture thus produced is then sterilized. The sterilization may require a somewhat longer period than that required for liquid media, as the heattransfer properties of the carrier materials tend to be poor. Usually,

however, a period of from three to five hours at a steam pressure of 15 to 20 pounds is sufllcient for batches of up to about 10 gallons, although When the sterilization step has been completed,

the mass is cooled to a temperature of between as which it is allowed to ferment in the presence of about25 and C. and then inoculated with an active culture of Eremothecium Ashbyii, after a sterile oxygen containing atmosphere such as air. During the fermentation, the medium is agitated either continuously or intermittently in order that the mold spores may be uniformly dis- 40 persed. When the fermentation is finished, the

substantially dry mass may, if desired, be used as such to supplement stock feeds of various types which are deficient with respect to their riboflavin and pantothenic acid contents. If desired,

however, the, substantially pure riboflavin can be obtained by extracting the solid fermented mass. for example, with hot water or with a suitable dilute acid such as dilute acetic acid. The riboflavin present in the extract may then be recovgrowth and riboflavin et d tion of the ered therefrom in accordance with any of several organism. I

A further object of my invention is to prepare a material high in B-complex vitamins including riboflavin, in a form particularly suitable for blending with animal feed material.

known methods, for example, by adsorption on a suitable adsorbent material such as fullers earth oradsorbent clay. The resulting adsorbate can then be treated in known manner with a suitable elution solvent, as for example, that described in U. S. Patent No. 2,343,254 g No. 2,006,699 of Supplee, to obtain the riboflavin in a substantially pure state.

' .a sugar such as glucose dissolved in yeast water or the aqueous residues obtained from ayeast fermentation sometimes called thin grain slopand containing dissolved glucose. Typical yeast fermentation residues which may be utlhzed are those resulting from corn, com-rye, corn-wheat.

or similar mashes. While the concentration of dissolved glucose may vary, I have found that nutrient solutions containing from between about 0.5 to 1.0% of a sugar such as glucose are quite satisfactory. Solutions having a lower sugar con- 1 centration than 0.5% are generally less desirable since larger volumes may be necessary to furnish the required proportion of glucose and thus frequently result in converting the solid medium on which the mold is grown into a sticky or sloppy intractable mass. Ordinarily nutrient solutions containing from about 0.5 to about 1.0% sugar when added in amounts corresponding to about 300350 parts by weight of solution for each 325-350'parts by weight of support or carrier employed will produce a high yield of riboflavin. The addition of the nutrient solution can be adjusted to contain only the quantity and kind of nutrients required to supplement the nutrient values existing in the carrier. It should be pointed out that since the aqueous yeast fermentation residues described above, have a comparatively low pH, it is usually desirable to adjust the acidity thereof to a value of around 6.0 to 6.5. This can readily be accomplished by the addition of dilute alkali to said residue prior to mixing with the carrier. In further regard to the expression nutrient solution, as used throughout the present disclosure and appended claim, it is to be strictly understood that such terminology is intended to cover any nutrient solution that has been previously known to support the growth of the fungus Eremothecium Ashbyii and in parj ticular, the solutions obtained by dissolving ,a sugar such as glucose, preferably in the amounts indicated above, in yeast water or aqueous residues from any yeast fermentation, or if desired, the sugar may be replaced in part by a metabolizable lipid as described in copending application, Serial No. 465,383, filed November 12, 1942, Patent No. 2,374,503.

The carrier or support used in the process of my invention may be in fluify, granular, shredded, flake or similar form. It must have a relatively large surface area per unit weight, it should not coalesce to become sticky or gummy throughout the fermentation period, it must remain porous and light during processing and no substances should be present or formed which specifically inhibit the fermentation or riboflavin production. Carriers found to be particularly suitable are those selected from the class consisting of oat hulls, corn bran, wheat bran and bone meal. These carriers contribute to the nutrient requirements .of the fermenting organism.

For optimum results, the container in which fermentation is to be carried out should be filled in such away with the carrier as to permit of such'ready mixing and air distribution as to satisfactorily supply the required aerobic conditions for growth and vitamin production of the orof Dale or U. S. Patentganism. Using a drum of the type described in the examples included hereinafter, I

have found that the proportion of carrier should not exceed about three-fourths of the total volume of said container. Otherwise, it becomes diflicult mass.

' The fermentation may be effected at temperatures varying from about 20 to 35 C. However.

it has been found preferable to employ temperaturesof between approximately 25- and 30 C. Temperatures within the latter range appeared to bring aboutv a favorable growth of the mold, resulting in a relatively short period of the order of several days for completion of the fermentation.

The container employed in carrying out the process of my invention may be of any suitable design. For the majority of purposes I prefer, however, to utilize a closed fermentation vessel containing a sterile oxygen containing gas such as air and adapted to--be rotated continuously or intermittently during the fermentation. In a. vessel of this type, thec'arrier to which the nutrient solution has been added and mold culture grown thereon are effectively created in an atmosphereof sterile .air, or the like, by merely revolving the vessel in a. manner such that the carrier travels part way up the sides of the vessel and then falls back to the bottom thereof. A variationof the above, by which effective aeration of the medium is obtained, embodies the use of a vessel equipped with entrance and exit ports for sterile oxygen containing gas such as air as well as a suitable sparger i. e. a perforated air inlet member to insure a substantially uniform distribution of the air through the solid medium. The exit port of such vessel should be sealed from the atmosphere to avoid contamination. This object can readily be accomplished for example, by the use of a suitable liquid seal or by inserting a sterile cotton plug in the exit port. With vessels of the latter type sterile air may be introduced at a'rate of from between about 0.045 cubic foot to about 9.5 cubic feet of air per hour per cubic foot of solid medium. As far as I have been able to determine, the introduction of air in amounts which are in excess of the aforesaid range result in only a negligible increase in the yield of riboflavin.

My invention may be more specifically illustrated by the data appearing in the tables below.

In Table I is shown the riboflavin production from nutrient solutions on various carriers when a partially filled container was agitated by rotation and aerated by diffusion through porous sterile plugs used as closures for the containers. In this series of tests, the nutrient medium was made up by adding 0.5% dextrose to an aqueous residue from the yeast fermentation of wheat and wheat flour mesh. The pH was adjusted to 6.1 with 10% NaOH solution and the mixture was used in the quantities indicated in the table to moisten the indicated quantity of carrier. The

media were placed in the rotating containers and autoclaved for three hours at 15 pounds steam pressure.- Each container was inoculated with 30 parts of a 48 hour 'old second generation flask culture of Eremothecium Ashbuii. The containers were rotated, continuously or intermit-' tently as indicated in the table, at a rate of 1.3 RPM. The bran was. thereby carried partway up the side of the container and fell back to the lower side. again at each revolution, serving to thoroughly mix and aerate the culture. After the indicated number of days. the culture medium acid in: thirty minutes. a: a-ayed for riboflavin with a photofluoromobservedthatcornbranandwheat whtchundertheconditionsoiusewillnotbecome sticky or glue-like but preferably remains porous and light 'during processing, and which contains no inhibitory substances. The yields of 5 riboflavin may vary widely depending upon the closeness with which the particular carrier used meets these desired characteristics. Some rather closely related carrier materials are decidedly less suitable than the preferred materials disclosed in Tables I and II and give substantially 3 lower yields than disclosed therein.

TABLI I Riboflavin production on solid base-Rotated container N 'lem Time 298g! t '0 Da ail-d1 our Other y tore lzedium Toe 1-215 24 5 1,000 Continuous rotation. res-21s 24 1 2,500 Do. TOE-4mm..- 2i 9 2,300 Ingrm i tient rotation four hours i 'ro 24 9 3,300 Da res-120 parts..... 24 7 L640 Do. W p 24 7 2,810 Do. Water-170 paris.-.-- 24 1 1,000 Do. res-110 24 1 1,000 Do. res-1:10 parts".-- 24 1o amt-2,000 Do.

1 Yeast lermeutation residue, e. g. thin grain slop.

In Table II is shown riboflavin production of positively aerated cultures. In this series or tests a container was used which had an inlet for compressed air in the top and small holes in the bottom for air outlet. Over the bottom of the can was placed a cotton pad and a paper cap. The carrier-nutrient compositions were placed in the container so as to occupy about three-fourths of the available space, and the container and contents were autoclaved for three hours. The sterilized medium was then inoculated with forty parts of a forty-eight hour old second generation flask of Eremothecium Aahbyii, and then sterilized, humidified air was passed through the culture at rates varying from 0.005 cubit foot per hour to 0.9 cubic foot per hour. The culture was agitated vigorously immediately after inoculation, and again each day to thoroughly distribute the spores through the medium.

After the indicated number of days, the media were removed, extracted and assayed for riboi'iavin.

so The various experimental conditions such as temperature, aeration rates, and methods of bringing the mold into contact with sterile oxygen containing gas may be subjected to numerous modifications without departing from the scope of my invention. It culture media other than those of the type specifically mentioned are util- Y ized, those skilled in the art may readily make the necessary adjustments to obtain the experimental conditions indicated above. I

My invention now having been described, what I claim is:

In the biochemical synthesis of riboflavin, the process which comprises impregnating a porous solid material in particulate form, selected from a class consisting of oat hulls, corn bran, wheat bran and bone meal, with an aqueous nutrient medium, having a sugar concentration of about 0.5-1.0% by weight, in quantity sufficient to moisten said carrier material and to form a liquid film over the surface of the particles thereof, sterilizing the mass, inoculating with an active TABLE II Riboflavin production on solid base-Stationary aerated container Carrier Nutrients added T De Ti Riggflavin, 0111 ['3- me rgram ture, o. Days fir dry 0111- marks Kind Parts Dextrose Other ture Medium Parts Wheat Bran--- 325 1.4 TGS -275 parts--- 24 7 soon-4,000 Continuous high air rate. 0.9 cu.

Do 325 1.4 TGS-275 parts 24 0 3,200 'Do'. Do 325 1.4 res-21s parts so 4 2,600 Do. Do an 1.5 TGS-300parts.- u a 1,100 Do. Do 325 1.5 TGS-300 parts"--. 24 0 3,300 Intgnaii/fint Low Arr rate 0.005

1 Yeast fermentation residue, e. g. thin grain slop.

It is to be understood, of course, that the above examples are merely illustrative and are to be in no way considered limitative. These examples show that the carrier selected must be one that culture of Eremothecium Aahbyii, maintaining the temperature of the inoculated mass within the range of about 20-35 C. and the pH within the range of about 6.0-6.5, supplying sterile air has a ame sur ace a pe unit weight and 7 to aerate the mass at the rate of from about 0005-95 cubic feet per hour per cubic feet of FOREIGN PATENTS 2122mm s zzzmazzar JEROME MARTIN. 5 OTHER REFERENCES REFERENCES CITED Industrial and Engineering Chemistry, Re-

ports on the chemical world today, December The following references are of record 1n the 943 5 file of this patent: Rafi'y et a1.: Comptes Rendus, Tome 205 (1937) UNITED STATES PATENTS 10 Pages 1005-1006- Rafly et aL: Comptes Rendus, Soc. de. Biologie Number Name Date (1937) pages 875-877.

1, 8 26 Wwyenaka s- 14, 1928 Guilliermond: Comptes Rendus, T. 200 (1935), 2,006,086 May J ne 2 1 pages 55 t 553, 2,291,009 Underkoifler July 28, 1942 15 Schopfer: Helvetica. 0111mm Acta, v. xxvu,

2,374,503 Rudert Apr. 24, 1945 pages 10 7 3 

